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Postharvest Biology and Technology 56 (2010) 138–146

Contents lists available at ScienceDirect

Postharvest Biology and Technology

journa l homepage: www.e lsev ier .com/ locate /postharvbio

thylene regulation of avocado ripening differs between seeded and seedless fruit

era Hershkovitza,b,∗, Haya Friedmana, Eliezer E. Goldschmidtb, Edna Pesisa

Department of Postharvest Science of Fresh Produce, The Volcani Center, P.O. Box 6, Bet Dagan 50250, IsraelThe Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel

r t i c l e i n f o

rticle history:eceived 3 November 2009ccepted 23 December 2009

eywords:old storagethylene receptor genesthylene biosynthesis genes-MCP

a b s t r a c t

We studied the contribution of the seed to avocado ripening, emphasizing its role in ethylene biosyn-thesis and response pathways. Transcription profiles of genes involved in ethylene biosynthesis (PaACO,PaACS1 and PaACS2) and action (PaETR, PaERS1 and PaCTR1) were studied in seeded and seedless avo-cado fruit during ripening at ambient and low temperatures and in response to exogenous ethylene and1-methylcyclopropene (1-MCP). Seedless mature fruit had a shorter preclimacteric lag, faster softening,and higher respiration during ambient temperature ripening than seeded ones. Advanced ripening inseedless fruit was accompanied by higher levels of PaACO and PaACS1 expression at harvest, and theselevels increased dramatically towards the climacteric peak. The expression of PaETR, PaERS1 and PaCTR1increased in parallel with the onset of the ethylene burst in seedless fruit, whereas PaETR increase pre-dominantly in seeded ones. Seedless fruit exhibited an earlier response to exogenous ethylene at theday of harvest, than seeded fruit. On day 1 after harvest, ethylene application elicited lower levels ofethylene in seeded than in seedless fruit, concomitantly with massive PaCTR1 augmentation. This sug-gests that the negative regulator PaCTR may moderate the effect of ethylene on seeded fruit. Cold storageinduced biosynthesis and regulatory genes in both seedless and seeded fruit relative to their levels atambient temperature. However, in the first and second weeks in cold storage, PaACO, PaACS1 and PaACS2expression levels were much higher in seedless than in seeded fruit, which could explain the higher lev-els of ethylene and accelerated softening of the seedless fruit in cold storage. Seeded and seedless fruit

responded similarly to ethylene or 1-MCP application prior to cold storage. Ethylene slightly inducedethylene production, but significantly increased CO2 output. 1-MCP equally and effectively delayed soft-ening, reduced ethylene and CO2 production and expression of genes involved in ethylene biosynthesisand ethylene action in seeded and seedless fruit. Both at ambient temperature and in cold storage res-piration was higher in seedless than seeded fruit. Our findings demonstrate that the seed is involved inregulation of ethylene responsiveness during ripening, and acts to delay climacteric in mature seeded fruit.

. Introduction

The avocado fruit (Persea americana Mill.) is a subtropi-al climacteric fruit, characterized by high postharvest increasef ethylene (Bower and Cutting, 1988). Expression of 1-minocyclopropane-1-carboxylic acid (ACC) synthase (ACS) geness generally differentially and tightly regulated by various develop-

ental, environmental and hormonal signals (Kende, 1993). Tracemounts of ACC and very low ACS activity were found in pre-limacteric avocado fruit; these increased markedly during thelimacteric rise and reached a maximum shortly before the ethy-

∗ Corresponding author at: Department of Postharvest Science of Fresh Produce,he Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel. Tel.: +972 3 9683619;ax: +972 3 9683622.

E-mail address: vhershko@agri.gov.il (V. Hershkovitz).

925-5214/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.postharvbio.2009.12.012

© 2010 Elsevier B.V. All rights reserved.

lene peak (Sitrit et al., 1986). ACC oxidase (ACO) activity in avocadoincreased markedly only at the ripening-associated upsurge ofethylene (Owino et al., 2002). It has been shown that low tem-peratures during storage stimulate ACO and ACS gene expressionin apples and pears (Tian et al., 2002; El-Sharkawy et al., 2003;Fonseca et al., 2005), and that chilling temperatures in the orchardcaused dramatic induction of avocado fruit ripening with parallelincreases in ethylene biosynthesis and expression of receptor genes(Hershkovitz et al., 2009b).

Ethylene is perceived by receptors encoded by a multigenefamily that includes, besides constitutively expressed members,some genes that are up-regulated during the onset of ripening

(Bleecker, 1999; Klee, 2002). CTR1, a key negative regulator of ethy-lene responses, acts downstream to the ethylene receptors, and inArabidopsis, AtCTR1 is constitutively expressed (Kieber et al., 1993).A multigene family of functional CTR1 genes is present in tomato;its members are differentially regulated by ethylene during the

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arious stages of development (Adams-Phillips et al., 2004). Forxample, LeCTR1 is up-regulated by ethylene during tomato fruitipening (Leclercq et al., 2002). Ethylene response-pathway ele-ents and their expression patterns are an important aspect of

he study of ripening in various fruit species (Owino et al., 2002;iersma et al., 2007).The seed has a major effect on avocado fruit development,

ainly with regard to fruit growth (Blumenfeld and Gazit, 1974).eedless fruit in avocado represent a type of stenospermocarpy,hereby fertilization occurs, but the seed aborts and fails toevelop, and such fruit are always smaller than seeded onesBlumenfeld and Gazit, 1974; Lovatt, 1990). Seed development isecessary for cell division within the grape berry mesocarp (Friendt al., 2009), and the presence of the seed delays the onset ofrape fruit maturation (Cawthon and Morris, 1982; Retamales etl., 1993). Our previous data suggest that in ‘Arad’ avocados, whichave a relatively large number of seedless fruit growing alongsideeeded ones (Lavi et al., 2000), the seed enhances ethylene sensi-ivity of the mesocarp tissue surrounding the seed during the lateipening process (Hershkovitz et al., 2009a).

Application of 1-methylcyclopropene (1-MCP) has beeneported to delay avocado ripening, as expressed in maintainingrmness, peel color, and reducing ethylene production and res-iration rates (Feng et al., 2000; Jeong et al., 2002; Hershkovitzt al., 2005). Treatment of avocado with 1-MCP prior to coldtorage also can reduce chilling injury, membrane permeability,olyphenol oxidase (PPO), peroxidise (POD) activities and embryoevelopment (Pesis et al., 2002; Woolf et al., 2005; Hershkovitz etl., 2005, 2009a).

The present study aimed to investigate the involvement ofhe seed in avocado ripening, by using the system of seeded andeedless fruit of ‘Arad’ fruit. The differences between seeded andeedless fruit in ripening, responses to ethylene and in expressionf ethylene biosynthesis and action related genes were examined atmbient temperatures and during cold storage, and also following-MCP application.

. Materials and methods

.1. Plant material and postharvest treatments

Seeded and seedless avocado (Persea americana Mill. cv. Arad)ruit were harvested at the same developmental stage and in accor-ance with commercial standards for dry weight, in January 2006nd January 2007, from the orchard of Kibbutz Ma’abarot in theentral Coastal Plain of Israel. In Experiment 1, seeded and seedlessruit were held at 20 ◦C for normal ripening. In Experiment 2, fruitere pretreated on harvest (E0) or on day 1 after harvest (E1) with

thylene at 10 �L L−1 or with 1-MCP at 150 nL L−1, each for 18 h at0 ◦C, while untreated control fruit were left in air for 18 h at 20 ◦C.0 fruit were kept at 20 ◦C, while E1 fruit were divided and partf them were kept at 20 ◦C and another part were transferred to aefrigerated chamber for cold storage at 5 ◦C for 4 weeks followedy two days at 20 ◦C. 1-MCP was generated from SmartFreshTM con-aining 0.14% (w/w) active ingredient (Rohm & Haas, Philadelphia,A, USA).

Fruit in Experiment 1 were sampled on days 0, 1, 4, 7, 9, 11 and3 at 20 ◦C; those in Experiment 2 were sampled immediately afterreatments, 1, 2, 3, and 4 weeks at 5 ◦C, and after 4 weeks at 5 ◦Collowed by two days at 20 ◦C. Fruit pulp internal to the blossomnd was frozen in liquid nitrogen, stored at −80 ◦C, and used for

NA preparation.

Fruit firmness (N) was determined on whole, unpeeled fruit withn LTCM electronic penetrometer (Chatillon, New York, NY, USA)ith a 6.5-mm conical probe. Penetrations of 12 mm were made at

wo equidistant points on the equatorial region of each fruit, at a

and Technology 56 (2010) 138–146 139

penetration rate of 3 mm s−1. The fruit were considered completelysoft or “ready to eat” when the average firmness value decreasedto 5–10 N.

2.2. Determination of ethylene and CO2 production

Individual fruit were sealed in 2 L glass jars and held at 20 ◦Cfor 1 h. Headspace gas samples were then withdrawn from eachjar with a 10 mL syringe. Ethylene and carbon dioxide contents inthe gas sample were determined by gas chromatography (GC) asdescribed by Pesis et al. (2002).

To determine ethylene and CO2 production during cold storage,1 g discs were cut from the inner pulp, close to the blossom end, pre-pared and sealed in a 50 mL Erlenmeyer flask with 1 mL of water onWhatman no. 1 filter paper. After 1 h of incubation at 20 ◦C, ethyleneand CO2 contents were determined by GC as described above.

2.3. RNA extraction and cDNA synthesis

Frozen avocado pulp was ground in liquid nitrogen with a mor-tar and pestle. Total RNA was extracted by means of the SV TotalRNA Isolation System (Promega, Madison, WI, USA), according tothe manufacturer’s instructions. Total RNA was analyzed by bothgel electrophoresis and NanoDrop-1000. First-strand cDNA wassynthesized with Reverse-iTTM 1st Strand Synthesis Kit (ABgene,Epson, UK), from 1 �g of total RNA that had been pretreated with1.5 units of RQ1 (Promega, Madison, WI, USA).

2.4. Quantitative real-time RT PCR

Quantitative real-time RT PCR (Q-RT-PCR) of PaACS1(AF500119), PaACS2 (AF500120), PaACO (M32692.1), PaETR(EU370699), PaERS1 (AF500121) and PaCTR1 (EU417962) tran-scripts accumulation was performed with a GENE 6000 instrument(Corbett Life Science, Sydney, Australia) using gene-specificprimers, according to Hershkovitz et al. (2009b). For all the genesstudied here, the optimal primer concentration was 200 nM. Reac-tions were carried out with 5 �L of the Absolute QPCR SYBR GreenROX Mix (ABgene, Epson, UK), according to the manufacturer’sinstructions. The Q-RT-PCR conditions were as follow: incubationfor 15 min at 95 ◦C, followed by 40 cycles of 95 ◦C for 15 s, 60 ◦C for20 s, and 72 ◦C for 20 s.

Harvest day in seeded fruit was designated as the calibrationpoint, set as 1, for relative expression levels, which were calculatedaccording to the comparative Ct method (Livak and Schmittgen,2001), with avocado PaGAPDH (GQ122209) used as an internal stan-dard.

Variations in quantitization were minimized by using two cDNAsynthesis reactions for each sample in separate Q-RT-PCR reac-tions for ethylene biosynthesis genes and two independent RNAextractions for ethylene response-pathway genes.

3. Results

3.1. Ethylene and CO2 production, firmness and gene expressionduring ripening at 20 ◦C

Ethylene production in seeded avocado fruit started to increaseon day 7 and reached a maximum value of 130 �L kg−1 h−1 on day11 after harvest; in seedless fruit it reached a maximum on day 9.

Seedless fruit had a shorter preclimacteric lag period, and ethyleneconcentrations at the climacteric peak reached 150 �L kg−1 h−1

(Fig. 1A). CO2 production in seedless fruit was significantly higherthan in seeded ones; it peaked on the fourth day preceding theethylene peak and fell to a minimum on day 11 (Fig. 1B). How-

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ver, the CO2 levels in seeded fruit rose significantly on day 11n parallel with the increase in ethylene production (Fig. 1A and). Seedless fruit softened rapidly and linearly, and were ready toat within nine days, whereas seeded fruit ripened much morelowly and completed their ripening thirteen days after harvestFig. 1C).

Transcript levels of PaACO, PaACS1 and PaACS2 genes were muchigher in seedless than in seeded fruit during the first four daysfter harvest, but on the seventh day after harvest there was a dra-atic increase in these gene levels in the seeded fruit (Fig. 2A–C)hich preceded the ethylene peak (Fig. 1A). The levels of PaACO,

aACS1 and PaACS2 transcripts remained high in both types ofruit even after they had softened completely, on days 9 and 11n seedless and seeded fruit, respectively (Fig. 2A–C). PaETR tran-cript expression was very low on the day of harvest in both seedednd seedless fruit and increased significantly, concomitantly withclimacteric peak of ethylene production (Fig. 3A vs. Fig. 1A). Onay 11 PaETR expression levels in seedless fruit decreased, in asso-iation with the reduction in ethylene production, whereas those

n seeded fruit were still high. Interestingly, one day after harvestaETR expression in seeded fruit increased, but declined later, andncreased again towards the climacteric peak (Fig. 3A). Expressionf PaERS1 and PaCTR1 in seedless fruit peaked on days 7 and 9,

ig. 1. Ethylene production (�L kg−1 h−1) (A), respiration (mg kg−1 h−1) (B) and firm-ess (N) (C) during storage at ambient temperature (20 ◦C) for seedless and seededvocado fruit, cv. Arad. Vertical lines represent SE for average of five different fruit.

Fig. 2. Relative gene expression of PaACO (A), PaACS1 (B) and PaACS2 (C) duringstorage at ambient temperature (20 ◦C) for seedless and seeded avocado fruit, cv.Arad. Vertical lines represent SE for an average of three replicates.

respectively, and both fell dramatically at the postclimacteric stageon day 11 (Fig. 3B and C). In contrast, in seeded fruit the expres-sion levels of PaERS1 and PaCTR1 were very low during the firstweek at 20 ◦C, but rose on days 9–11 in parallel with the increasein ethylene production (Fig. 3B and C vs. Fig. 1A). Also PaCTR1expression in seeded fruit increased slightly one day after harvest,declined later, and increased again on approaching the climactericpeak (Fig. 3C).

3.2. Effect on ripening of exogenous ethylene pretreatments givenimmediately or 24 h after harvest

Application of ethylene to seeded fruit on the day of harvest(E0) did not advance the ethylene peak, whereas in seedless onesthe ethylene peak appeared earlier than in the controls (Fig. 4Sand SL). On the other hand, ethylene application 24 h after harvest(E1) advanced the ethylene peak dramatically in both seeded and

seedless fruit: it appeared after five days in both types. However,in seedless fruit this ethylene peak was significantly higher than incontrol fruit, whereas in seeded fruit it was significantly lower thanin seeded controls (Fig. 4S and SL).

ology and Technology 56 (2010) 138–146 141

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E0 slightly induced PaACO transcript expression only in seededruit, but did not influence transcription levels of PaACS1 andaACS2 in either seeded or seedless fruit. On the other hand, E0 sig-ificantly induced the expression levels of PaETR and PaERS1 in botheeded and seedless fruit. In addition, there was dramatic induc-ion of PaCTR1, but only in seeded fruit (Fig. 5 E0-S vs. E0-SL). Onhe contrary, E1 induced PaACO, PaACS1, PaACS2 and PaETR tran-cript levels in both seeded and seedless fruit, but this inductionas 10–30-fold higher in seedless ones. Also, PaERS1 and PaCTR1

ene expression increased in response to ethylene application on1, but only in seeded fruit (Fig. 5E1-S vs. E1-SL).

.3. Effect of ethylene or 1-MCP pretreatments on production ofthylene and CO2 and on firmness during cold storage

Ethylene treatment prior to cold storage at 5 ◦C advanced ethy-ene production during storage by 1 week compared with theirontrols, in both seeded and seedless fruit: ethylene was detected

ig. 3. Relative expression of PaETR (A), PaERS1 (B) and PaCTR1 (C) during storage atmbient temperature (20 ◦C) for seedless and seeded avocado fruit, cv. Arad. Verticalines represent SE for an average of three replicates.

−1 ◦

Fig. 4. Effects of exogenous ethylene application (10 �L L , 18 h at 20 C) immedi-ately after harvest (E0) or 24 h after harvest (E1) on ethylene production in seeded(S) and seedless (SL) avocadoes during storage at 20 ◦C. Vertical lines represent SEfor an average of five different fruit.

after 1 week in ethylene-treated fruit, compared with 2 weeks inthe controls (Table 1). The CO2 production levels in both fruit typesincreased significantly in response to ethylene application, and dur-ing the first 2 weeks at 5 ◦C were higher in ethylene-treated fruitthan in the controls. Following storage when fruit were removed toshelf-life at 20 ◦C, a significant increase in ethylene was observed inboth seeded and seedless fruit. 1-MCP treatment completely inhib-ited ethylene production for 3 weeks in seeded fruit compared with2 weeks in seedless ones. CO2 production in 1-MCP treated fruit,both seeded and seedless, was lower during all the period of coldstorage, than in their controls (Table 1).

Seedless and seeded fruit differed also in their softening rate atcold storage. While seedless fruit already became edible soft duringcold storage of 3 weeks this stage occurred in seeded fruit only after4 weeks in cold storage plus two days shelf-life. In addition, ethy-lene enhanced softening during storage of only seedless fruit, andthis correlated with higher induction of ethylene in seedless fruit(Table 1). Both seeded and seedless avocadoes treated with1-MCPremained firm, at 116 N and 110 N, respectively, during cold stor-age and began to soften only after removal to shelf-life conditions(Fig. 6S and SL).

3.4. Effect of ethylene or 1-MCP pretreatment on expression ofPaACO, PaACS1 and PaACS2 genes during cold storage

In seeded fruit the expression patterns of all three genes were

similar, but the increase in PaACS1 transcript levels was more dra-matic than that for PaACS2 transcripts (Fig. 7A-S, C-S and E-S). Inboth seedless and seeded fruit PaACO transcripts gradually accu-mulated during cold storage, and reached their highest levels after

142 V. Hershkovitz et al. / Postharvest Biology and Technology 56 (2010) 138–146

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ig. 5. Effects of exogenous ethylene treatment (10 �L L−1, 18 h at 20 ◦C) immediateaERS1, PaETR and PaCTR1 transcript expression in mature seeded (E0-S, E1-S) and

ruit were transferred to shelf-life conditions at 20 ◦C (Fig. 7A-S and-SL). On the other hand the levels of PaACS1 and PaACS2 tran-cripts increased gradually during cold storage and shelf-life onlyor seeded fruit (Fig. 7C-S and E-S), whereas in seedless fruit theyeached their maximum levels after only 1 week at 5 ◦C (Fig. 7D-SLnd F-SL). Moreover, PaACS2 levels were higher in seedless than ineeded fruit (Fig. 7F-SL vs. E-S).

In both seeded and seedless fruit, exogenous ethylene applica-ion prior to cold storage significantly induced PaACO expressionevels compared with those in the controls in the first week at 5 ◦C

Fig. 7A-S and B-SL). In seeded fruit ethylene also increased thexpression of PaACS2 in the first week at 5 ◦C (Fig. 7E-S). 1-MCPreatment decreased transcript levels of all three genes in both fruitypes during cold storage at 5 ◦C (Fig. 7). Following 1-MCP treat-

ent, removal of fruit to shelf-life conditions caused induction of

able 1ffect of ethylene on the day after harvest (E1) or 1-MCP application on ethylene and CO2

Fruit type Fruit/Trt. Storage period

Weeks at 5 ◦C

End of Trt. 1

SeededEthylene (nl g−1 h−1) S-Cont. nda nd

S-E1 nd 1.1 ± 1.1S-MCP nd nd

CO2 (�g g−1 h−1) S-Cont. 101.9 ± 4.5b 122.7 ± 6.3bS-E1 159.5 ± 13.5a 164.9 ± 13.7aS-MCP 62.2 ± 6.3c 69.7 ± 6.3c

SeedlessEthylene (nl g−1 h−1) SL-Cont. nd nd

SL-E1 nd 2.6 ± 0.4SL-MCP nd nd

CO2 (�g g−1 h−1) SL-Cont. 165.7 ± 14.1b 147.6 ± 7.2bSL-E1 186.3 ± 13.6a 167.3 ± 6.6aSL-MCP 94.1 ± 2.1c 94.0 ± 3.2c

thylene and CO2 levels were measured immediately after treatments and after storage fa nd: non-detectable.b Values within a column in each group followed by the same letter are not significantl

r harvest (E0-S, E0-SL) or 24 h after harvest (E1-S, E1-SL), on PaACO, PaACS1, PaACS2,ss (E0-SL, E1-SL) avocado fruit. Vertical lines represent SE for three replicates.

PaACS1 and PaACS2 in seeded fruit (Fig. 7C-S and E-S), whereas inseedless fruit there was induction of PaACO (Fig. 7B-SL). Gener-ally, in both seeded and seedless fruit PaACO, PaACS1 and PaACS2expression levels increased in 1-MCP treated fruit during the coldstorage.

3.5. Effect of ethylene or 1-MCP pretreatments on expression ofPaETR, PaERS1 and PaCTR1 genes during cold storage

Under cold storage PaETR transcript expression followed the

same trend in both seeded and seedless fruit: in control fruit ofboth types it was induced by cold, up to 4 weeks, and then declinedduring shelf-life (Fig. 8A-S and B-SL). In seeded fruit PaERS1 expres-sion remained at a basal level during cold storage, but the levelsincreased only after 4 weeks at 5 ◦C and after transfer to 20 ◦C

production in seeded (S) and seedless (SL) avocado cv. Arad.

Days 20 ◦C

2 3 4 2

1.3 ± 1.1ab 8.4 ± 0.6a 15.0 ± 0.4b 24.5 ± 0.9a2.9 ± 1.6a 8.9 ± 3.2a 29.1 ± 10.1a 27.2 ± 3.7and nd 9.6 ± 0.6b 19.9 ± 2.8b

112.3 ± 9.4b 154.1 ± 9.8b 201.1 ± 11.7b 280.5 ± 8.4a163.3 ± 13.2a 188.9 ± 8.0a 169.9 ± 14.8a 215.8 ± 5.6b68.3 ± 2.4c 79.7 ± 2.9c 108.5 ± 12.0c 200.0 ± 15.3c

2.3 ± 0.2b 5.6 ± 0.1b 14.2 ± 1.6b 24.4 ± 4.2b4.7 ± 0.7a 8.8 ± 1.4a 18.9 ± 1.2b 27.8 ± 3.6b0.1 ± 0.1c 0.1 ± 0.0c 14.9 ± 2.0a 42.1 ± 3.7a

165.6 ± 12.3b 192.7 ± 14.0a 205.0 ± 13.6a 207.9 ± 8.1b196.5 ± 11.2a 165.9 ± 6.6a 221.2 ± 12.5a 245.2 ± 9.2a113.5 ± 6.2c 119.1 ± 8.2c 144.8 ± 6.5b 199.4 ± 5.1b

or 1, 2, 3 and 4 weeks at 5 ◦C followed by two days at 20 ◦C.

y different according to Tukey’s test (P < 0.05) (ethylene, n = 4; respiration, n = 4).

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ig. 6. Effects of ethylene (10 �L L−1) or 1-MCP (150 nL L−1) on firmness of seededS) and seedless (SL) avocado fruit, cv. Arad, during 4 weeks at 5 ◦C following sixays at 20 ◦C. Vertical lines represent SE for five fruit.

Fig. 8C-S). In seedless fruit PaERS1 transcript expression patternsere similar to those of PaETR, with a peak after 4 weeks in cold

torage, followed by a decline after transfer to 20 ◦C (Fig. 8B-SL and-SL). PaCTR1 expression remained at low levels in both seeded and

eedless fruit during cold storage; it then increased 5-fold duringhelf-life at 20 ◦C (Fig. 8E-S and F-SL). Exogenous ethylene appli-ation prior to cold storage caused small changes in PaETR genexpression in both fruit types during cold storage, but dramaticallynduced the expression of this gene in seeded fruit during shelf-lifeFig. 8A-S and B-SL). Ethylene pretreatment significantly increasedaERS1 expression levels in seeded fruit during the first 3 weeksn cold storage, and induction was lower in seedless fruit (Fig. 8C-Snd D-SL). Induction of PaCTR1 by ethylene occurred only in seededruit, and was already observed after 1 week at 5 ◦C (Fig. 8E-S). 1-

CP treatment significantly decreased transcript levels of all threeenes in both fruit types during the entire cold storage periodFig. 8). After transfer of 1-MCP-treated fruit to shelf-life conditions,aETR expression increased only in seedless fruit in comparisonith control and ethylene-treated ones (Fig. 8B-SL).

. Discussion

.1. Delay of avocado ripening by the seed

The seed might have a strong effect on fruit growth and its con-ribution to fruit ripening has rarely been investigated. In grapes,rowth of normal seeded grape berries is characterized by bothell division and cell expansion (Friend et al., 2009), and in the

and Technology 56 (2010) 138–146 143

presence of seeds, fruit maturation can be delayed (Cawthon andMorris, 1982; Retamales et al., 1993). In contrast, parthenocarpicmelon fruit, which had only empty seed coats, exhibited a delay inclimacteric ethylene production (Kato et al., 2002). In our presentstudy we demonstrated that seedless fruit had a shorter precli-macteric period, preceding the onset of climacteric production ofCO2 and ethylene, accompany with accelerated softening and geneexpression, than seeded fruit (Figs. 1–3). This suggested that theavocado seed most likely delays ripening processes. It is plausible tohypothesize that seedless avocadoes progress to ripening becausecell division stops earlier in seedless than in seeded fruit on thetree.

The present study showed that respiration rates were higherin seedless fruit than in seeded ones during ripening at ambi-ent temperature (Fig. 1B) or at 5 ◦C (Table 1). High respirationrates in avocado fruit are associated with increased ability to pro-duce abscisic acid (ABA), particularly during the climacteric phase(Adato et al., 1976). Richings et al. (2000) showed that small fruit of‘Hass’ avocado had higher respiration rates, higher ABA and lowerindoleacetic acid (IAA) contents than larger fruit.

It is well documented, that ethylene production in plant tissueis regulated by the activity levels of both ACS and ACO (Bleeckerand Kende, 2000). The reason for earlier ethylene production inseedless than in seeded fruit, is probably because there are highertranscript levels of PaACO and PaACS1, already on the day of harvest.In the present study these high levels in seedless fruit also persistedon the first day at 20 ◦C, and increased dramatically toward theclimacteric peak (Fig. 2A and B). In contrast, PaACO, PaACS1, PaACS2transcript levels in seeded fruit were very low on the day of harvest,but increased markedly in association with climacteric ethyleneproduction (Fig. 1A vs. Fig. 2A–C). Thus, the present study confirmsprevious observations of detectable expression of avocado ACS andACO genes, but very low activity of ACS and ACO at harvest, andincreases in these activities with the onset of the climacteric riseduring avocado ripening (Owino et al., 2002). The inability of mostavocado varieties to produce ethylene as long as they are attachedto the tree results mainly from repression of ACS activity (Sitrit etal., 1986).

Compared with the high induction of ethylene biosynthesisgenes seen in seedless fruit, the ethylene response genes exhib-ited low transcripts levels in both seeded and seedless fruit uponharvest (Fig. 3 vs. Fig. 2); only PaETR transcript accumulated in par-allel with ethylene production in both seeded and seedless fruit(Fig. 3A), as we have already shown (Hershkovitz et al., 2009a).In seedless fruit, PaERS1 and PaCTR1 transcript levels were muchhigher than in seeded ones, and their levels increased correspond-ing with the climacteric rise in ethylene production. This suggestsa role for the seed in inhibiting the induction of ethylene responsegenes. Increases in ethylene response genes in parallel to ethyleneproduction have been reported in avocado and other fruit, includ-ing tomato NR (LeETR3), LeETR4, LeETR5 (Klee, 2002), pear PcETR1,PcERS1 and PcCTR1 (El-Sharkawy et al., 2003), plum Ps-ETR1 andPsERS1 (El-Sharkawy et al., 2007), avocado PaERS1 (Owino et al.,2002), PaETR and PaERS1 (Hershkovitz et al., 2009b), and appleMdERS1, MdETR2 (Wiersma et al., 2007) and MdCTR1 (Dal Cin etal., 2008). The increase in transcript expression in parallel with theethylene climacteric rise, supports the hypothesis that these ele-ments play an important role in moderating the ethylene response(Klee and Tieman, 2002).

4.2. Avocado ripening in response to exogenous ethylene at

The intact avocado fruit does not respond to exogenous ethy-lene immediately after harvest. Starrett and Laties (1991) showedthat application of short (24 h) pulses of exogenous ethylene or

144 V. Hershkovitz et al. / Postharvest Biology and Technology 56 (2010) 138–146

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ig. 7. Effect of ethylene (10 �L L−1) or 1-MCP (150 nL L−1) on relative expression ofnd seedless avocado fruit during 4 weeks at 5 ◦C followed by two days of shelf-life

ropylene to preclimacteric avocado did not cause immediate onsetf ripening, but did stimulate some biochemical and molecularhanges. As is evident from Fig. 4, only ethylene that was applied4 h after harvest (E1) was effective in advancing climacteric ethy-

ene production in either fruit type. However, seeded E1-treatedruit produced lower amounts of ethylene (Fig. 4S) in comparisonith climacteric ethylene production and that produced by seed-

ess E1-treated fruit. Reduction in ethylene levels due to ethylenepplication has been shown before in citrus (Riov and Yang, 1982)nd avocado (Zauberman et al., 1988). This effect paralleled anncrease in PaCTR1 expression, which occurred only in seeded fruitFig. 5). In contrast, in seedless fruit there was stimulation of ethy-ene production and no induction of PaCTR1 (Fig. 4SL vs. Fig. 5E1-SL).igher levels of PaCTR1 in seeded fruit might be responsible for

heir lower PaACO and PaACS levels than in seedless fruit, and mightccount for the lower levels of ethylene in this type of fruit. It shoulde noted that, during the preclimacteric stage, PaCTR1 transcript

evels were dramatically induced by exogenous ethylene only in

eeded fruit (Fig. 5E0-S), while endogenous climacteric ethyleneid induce it only in seedless fruit (Fig. 3C). This seems to suggesthat dynamic changes occur in the fruit after harvest which lead toifferent responses to ethylene of seeded and seedless fruit duringipening.

(seeded A-S, or seedless B-SL), PaACS1 (C-S, D-SL) and PaACS2 (E-S, F-SL) in seededcal lines represent SE for three replicates.

4.3. Effects of exogenous ethylene or 1-MCP and cold storage

Cold storage induced expression of genes for ethylene biosyn-thesis and ethylene action in both seedless and seeded fruit,compared with their levels at ambient temperature. However, inthe first and second weeks in cold storage the expression levelsof PaACO, PaACS1 and PaACS2 were much higher in seedless fruitthan in seeded ones, which could account for the higher levelsof ethylene in the second week and the consequent acceleratedsoftening of the seedless fruit in cold storage. Previously, it hasbeen reported that chilling stress, while the fruit were on the tree,caused dramatic induction of avocado fruit ripening and parallelincreases in expression of ethylene biosynthesis and receptor genes(Hershkovitz et al., 2009b). In apples and pears low temperaturesduring storage stimulate expression of ACO and ACS genes (Tian etal., 2002; El-Sharkawy et al., 2003; Fonseca et al., 2005). Similarly,cold storage induces ripening in ‘Stony Hard’ peaches, which arecharacterized by absence of ethylene production during ripening,

and in winter pears. This induction included stimulation of ACStranscription, ethylene production and softening (El-Sharkawy etal., 2003; Begheldo et al., 2008).

Application of ethylene prior to cold storage caused both seededand seedless fruit to respond similarly and to exhibit small induc-

V. Hershkovitz et al. / Postharvest Biology and Technology 56 (2010) 138–146 145

F vels ofa s repr

tptmmwehsaa

eidgivHci(sk

ig. 8. Effect of ethylene (10 �L L−1) or 1-MCP (150 nL L−1) on relative expression levocado fruit during 4 weeks at 5 ◦C followed by two days of shelf-life. Vertical line

ions in ethylene production, but significant increases in CO2roduction. However, up-regulation of the expression of biosyn-hesis genes and, especially, of ethylene response genes occurred

ainly in seeded fruit. Similar up-regulating effects of cold treat-ent and of exogenous ethylene treatment on PcERS1 expressionere found in pear (El-Sharkawy et al., 2003), which indicates that

thylene receptor transcription is positively controlled by ethylene,owever the effect on the levels of the receptors is still not clear,ince recently it has been demonstrated in tomato that ethylenepplication caused degradation of ethylene receptors (Kevany etl., 2007).

In the present study, application of 1-MCP induced no differ-nces between seedless and seeded fruit and it was equally effectiven the two fruit types, in delaying softening, ethylene and CO2 pro-uction during cold storage, and in down-regulating expression ofenes involved in ethylene biosynthesis and ethylene action. Sim-lar effects of 1-MCP in delaying ripening have been observed inarious avocado cultivars (Jeong et al., 2002; Pesis et al., 2002;ershkovitz et al., 2005, 2009a; Woolf et al., 2005) and in other

limacteric fruit (Watkins, 2006). 1-MCP treatment also inhib-ted ethylene-induced mesocarp discoloration during cold storagePesis et al., 2002; Hershkovitz et al., 2009a). Analogous suppres-ion by 1-MCP of receptor expression has been shown in apple andiwifruit during ripening at ambient temperature (Dal Cin et al.,

PaETR (A-S, B-SL), PaERS1 (C-S, D-SL) and PaCTR1 (E-S, F-SL) in seeded and seedlessesent SE for three replicates.

2006; Tatsuki and Endo, 2006; Yin et al., 2008). Similar behavior ofCTR1 after 1-MCP treatment has been observed in pear ripening at20 ◦C (El-Sharkawy et al., 2003).

The present results provide evidence for the involvement of theavocado seed in delaying fruit ripening processes. Firstly, seedlessfruit ripened more quickly, both at ambient temperature and in coldstorage, than seeded ones. The faster ripening was evident by anearly onset of climacteric ethylene production, significantly higherrespiration rates, and faster softening. Secondly, clear differenceswere observed between seeded and seedless fruit in the mRNAaccumulation patterns of the PaCTR1 gene in response to endoge-nous or exogenous ethylene; PaCTR1 was dramatically induced byexogenous ethylene only in seeded fruit, but endogenous ethyleneinduced it in seedless fruit. Thirdly, seedless fruit exhibited an earlyresponse to exogenous ethylene on the day of harvest, whereasseeded fruit did not. Taken together, these findings support thehypothesis that the seed contributes to avocado fruit ripening.

Acknowledgements

The authors would like to thank Oleg Feygenberg and Rosa Ben-Arie for their excellent technical assistance, and Rohm and HaasItalia Srl for the supply of 1-MCP. Contribution from ARO, the Vol-cani Center, P.O. Box 6, Bet Dagan 50250, Israel, no. 564/09.

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